The importance of being able to quickly and accurately determine the presence of Neisseria bacteria, particularly Neisseria gonorrhoeae, is well-appreciated. Conventional tests for detecting the presence of organisms such as N. gonorrhoeae require the preparation of bacteria cultures or the use of serological methods. Such tests, however, have well-recognized limitations. See, for example, the publication "International Symposium on Gonorrhea," B. B. Diena, Ed., a collection of papers presented at the October, 1973 International Symposium on Gonorrhea sponsored by the Health Protection Branch, Health and Welfare Canada, Ottawa, especially at page 34 et seq.
A relatively simple and quick enzymatic test for the presence of Neisseria bacteria is disclosed in the related patent application cited above entitled "Detecting Neisseria Bacteria". That test is based upon an enzyme assay for the enzyme, 1,2-propanediol dehydrogenase, which enzyme is specific to Neisseria bacteria. Thus, an enzyme was found in Neisseria which oxidizes 1,2-propanediol and reduces nicotinamide-ademine-dinucleotide (NAD) to NADH. The composition and structure of that enzyme are not fully understood nor has any identification therefor been found in the literature. Accordingly, its capability for oxidizing 1,2-propanediol and reducing NAD led to the adoption of the term 1,2-propanediol dehydrogenase therefor. That designation will be used throughout this specification.
In another related application cited above entitled "Detection of Neisseria Bacteria by Immunoassay" is disclosed the use of antibodies to inhibit the activity of the enzyme 1,2-propanediol dehydrogenase. And in yet another application cited above entitled "Immunoassay of Neisseria Bacteria Via (NH.sub.4).sub.2 SO.sub.4 Precipitation" is disclosed a modification of that method wherein (NH.sub.4).sub.2 SO.sub.4 precipitation of the antigen-antibody complex acts to concentrate the enzyme, remove interfering materials, and thereby improve the speed and precision of the assay.
In another related application cited above entitled "Immunological Detection of Neisseria Bacteria Via Labelled Antibodies," is disclosed a method for detecting N. gonorrhoeae cells that uses a radiolabelled antibody specific to the enzyme in two particular embodiments of radioimmunoassay techniques.
The instant disclosure is concerned generally with a process for detecting the presence of Neisseria bacteria by means of a radioimmunoassay (RIA) technique. It is particularly useful in detecting N. gonorrhoeae.
The reaction between an antibody and its homologous antigen is recognized as a specific biochemical reaction. In many instances, this reaction occurs even when the antigen is modified by the addition of a radioactive tracer. The antibody-antigen complexes then formed are radioactive and by various known means can be separated from uncomplexed reactants. As the measurement of radioactivity is a known and sensitive procedure, a quantitative assay for any protein to which a specific antibody is available is possible. In general such an assay requires antibody, purified antigen, and radiolabelled antigen. A known amount of antibody is mixed with a known amount of radiolabelled antigen and, after a suitable reaction time, the radioactive antibody-antigen complexes are separated from the uncomplexed radiolabelled antigen. This separation often occurs via a precipitation reaction that results when an antiglobulin antiserum is added which is specific for the gamma globulin in which the first antibody was prepared. The precipitate is collected by centrifugation and its radioactivity or the radioactivity in the supernatant solution is determined. Using the above procedure, the concentration of antigen can be determined by use of a standard reference curve. The curve is generated by mixing known amounts of antibody and labelled antigen with varying amounts of unlabeled antigen. In the reaction that occurs between the antibody and the two forms of antigen, the amount of radioactivity in the antibody-antigen complex is directly related to the concentration of unlabelled antigen in the reaction mixture. This occurs because the two forms of antigen are competing for the same, limiting amount of antibody. The amount of antigen in an unknown is determined by mixing a sample of the unknown with known amounts of antibody and labelled antigen. The radioactivity in the antibody-antigen complex is then related to the standard curve prepared with known amounts of unlabelled antigens.
In summary, a competition reaction between labelled antigen and unlabelled antigen for a limiting amount of antibody is established and the amount of radioactivity in the resulting antibody-antigen complexes is directly related to the concentration of unlabelled antigen in the starting reaction mixture.